Electrophoresis is widely used for fractionation of a variety of biomolecules, including DNA species, proteins, peptides, and derivatized amino acids. One electrophoretic technique which allows rapid, high-resolution separation is capillary electrophoresis (CE) (Grossman and Colburn, 1992). Typically, CE employs fused silica capillary tubes whose inner diameters are between about 10-200 microns, and which can range in length between about 5-100 cm or more.
One use for CE that has received much attention has been in the separation and identification of DNA sequencing fragments. Such separations have been carried out previously using slab gel configurations requiring painstaking preparation of crosslinked polyacrylamide matrices between glass plates. More recently, methods have been described for carrying out such separations by CE, providing the advantages of shorter separation times, reduced sample sizes, potential automation of sample loading, and potentially higher resolution of sample peaks.
Guttman et al. (1990) have reported single-base separation of polynucleotides containing on the order of 160 bases by CE using crosslinked polyacrylamide gels containing 3-6% T and 5% C.
Cohen et al. (1990) have demonstrated use of CE with a crosslinked polyacrylamide gel (3% T, 5% C) to separate DNA sequencing fragments differing in length by a single base from 18 to about 330 bases in total length.
Swerdlow et al. (1990a) have compared the separation of DNA sequencing fragments achieved by CE with that achieved by slab gel electrophoresis using identical crosslinked polyacrylamide matrices (6% T, 5% C). The separations afforded by CE were said to be 3-fold faster and to provide 2.4-fold better resolution and 5-fold better separation efficiency than provided by a conventional slab gel configuration.
Although crosslinked polyacrylamide matrices such as above have been shown to be useful in DNA sequencing analysis, certain limitations have remained. One limitation has been that bubbles can form in the polyacrylamide matrix during polymerization in the capillary tube, compromising peak resolution and necessitating rejection of some acrylamide-filled tubes following polymerization (Swerdlow, 1990a,b).
Another limitation has been the formation of bubbles near the injection end of the capillary tube during electrophoresis of the sample (Swerdlow, 1990b).
A third limitation has been that at high voltages, electroosmosis can occur, leading to extrusion of the gel matrix from the tube. To counter such extrusion, crosslinked matrices have been covalently attached to the inside wall of the tube (Karger et al., 1989). However, such covalent linkage can lead to the formation of voids in the matrix due to contraction during polymerization or electrophoresis (Grossman et al., 1992, at pp. 140-142).
A fourth limitation, in DNA sequence analysis, has been the fouling of the capillary inlet by the sequencing template. Accumulation of a large, essentially immobile template at the inlet can limit the degree of resolution achievable with subsequently loaded samples, thereby limiting use of the capillary to a few uses at most.
Another limitation has been that, when polymerization of the matrix is carried out in the capillary tube, the polymerization procedure must be performed individually for each tube and typically requires a significant delay (e.g., overnight polymerization) before the capillary can be used for electrophoresis.
Linear (non-crosslinked) polyacrylamide matrices have also been found useful in the separation of DNA fragments. Heiger et al. (1990) have shown that linear polyacrylamide matrices containing 6, 9, 12% T were useful in the separation by CE of restriction fragments ranging in size from about 75-12,000 basepairs in length (non-denaturing conditions), and further, that a higher % T (e.g., 9% T) was useful in resolving, under denaturing conditions, a mixture of polydeoxyadenylate fragments ranging from 40-60 bases in length. The authors suggested that polymerization of the polymer matrix be performed inside the capillary tube to obtain high viscosity and to minimize the difficulties of handling viscous solutions.
Sudor et al. (1991) reported separation of DNA fragments using linear polyacrylamide solutions containing 3-10% T (weight percent of total acrylamide) and 7M urea. Polyacrylamide-filled capillary tubes were prepared by forming the polyacrylamide solution outside of the tube and then forcing the polymerized solution into the tube by syringe, while taking care not to break the syringe due to excessive pressure. Comparison of CE separations performed with solutions containing 3, 5, and 10% T showed that 10% T gave the best resolution of oligonucleotide test fragments (poly-dC) 10 to 36 bases in length.
More recently, Mathies, Huang, and coworkers (Huang et al., 1992a,b,c; Mathies et al., 1992) have described a linear polyacrylamide matrix (9% T containing 7M urea) for DNA sequence analysis by CE. The matrix is said to typically allow sequencing of up to 300-350 bases per capillary (Huang et al., 1992b), and as high as 500 bases beyond the primer (Huang et al., 1992a). The highly viscous matrix is polymerized in the capillary tube and is said to be physically stable, allowing multiple (e.g., three or four) sample injections.
Ideally, a matrix for use in separating DNA sequencing fragments should (i) provide single-base resolution for DNA sequencing fragments of 300 bases in length, preferably 500 bases in length, or greater, and (ii) have a sufficiently low viscosity to allow rapid filling and re-filling of the capillary tube.